EP0355981A2 - Procédé de production de l'endonucléase de restriction et de la méthylase Nla IV - Google Patents

Procédé de production de l'endonucléase de restriction et de la méthylase Nla IV Download PDF

Info

Publication number: EP0355981A2
Authority: EP; European Patent Office
Prior art keywords: nlaiv; dna; gene; restriction endonuclease; clones
Prior art date: 1988-07-28
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.): Ceased

Application number

EP89307206A

Other languages

German (de)

English (en)

Other versions

EP0355981A3 (fr

Inventor

Richard D. Morgan

Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)

New England Biolabs Inc

Original Assignee

New England Biolabs Inc

Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)

1988-07-28

Filing date

1989-07-17

Publication date

1990-02-28

1989-07-17 Application filed by New England Biolabs Inc filed Critical New England Biolabs Inc

1990-02-28 Publication of EP0355981A2 publication Critical patent/EP0355981A2/fr

1990-03-07 Publication of EP0355981A3 publication Critical patent/EP0355981A3/fr

Status Ceased legal-status Critical Current

Links

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Classifications

- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
- C12N9/1007—Methyltransferases (general) (2.1.1.)

Definitions

the present invention relates to clones for the NlaIV restriction endonuclease and modification methylase, and to the production of these enzymes from the clones.
Restriction endonucleases are a class of enzymes that occur naturally in bacteria. When they are purified away from other contaminating bacterial components, zestriction endonucleases can be used in the laboratory to break DNA molecules into precise fragments. This property enables DNA molecules to be uniquely identified and to be fractionated into their constituent genes. Restriction endonucleases have proved to be indispensable tools in modern genetic research. They are the biochemical 'scissors' by means of which genetic engineering and analysis is performed.
Restriction endonucleases act by recognizing and binding to particular sequences of nucleotides (the 'recognition sequence') along the DNA molecule. Once bound, they cleave the molecule within, or to one side of, the sequence. Different restriction endonucleases have affinity for different recognition sequences. Over one hundred different restriction endonucleases have been identified among many hundreds of bacterial species that have been examined to date.
Bacteria usually possess only a small number of restriction endonucleases per species.
the endonucleases are named according to the bacteria from which they are derived.
the species Haemophilus aegyptius for example synthesizes 3 different restriction endonucleases, named HaeI, HaeII and HaeIII.
These enzymes recognize and cleave the sequences (AT)GGCC(AT), PuGCGCPy and GGCC respectively.
Escherichia coli RY13 synthesizes only one enzyme, EcoRI, which recognizes the sequence GAATTC.
restriction endonucleases play a protective role in the welfare of the bacterial cell. They enable bacteria to resist infection by foreign DNA molecules like viruses and plasmids that would otherwise destroy or parasitize them. They impart resistance by binding to infecting DNA molecule and cleaving them in each place that the recognition sequence occurs. The disintegration that results inactivates many of the infecting genes and renders the DNA susceptible to further degradation by exonucleases.
a second component of bacterial protective systems are the modification methylases. These enzymes are complementary to restriction endonucleases and they provide the means by which bacteria are able to protect their own DNA and distinguish it from foreign, infecting DNA. Modification methylases recognize and bind to the same nucleotide recognition sequence as the corresponding restriction endonuclease, but instead of breaking the DNA, they chemically modify one or other of the nucleotides within the sequence by the addition of a methyl group. Following methylation, the recognition sequence is no longer bound or cleaved by the restriction endonuclease.
the DNA of a bacterial cell is always fully modified, by virtue of the activity of its modification methylase and it is therefore completely insensitive to the presence of the endogenous restriction endonuclease. It is only unmodified, and therefore identifiably foreign, DNA that is sensitive to restriction endonuclease recognition and attack.
the key to isolating clones of restriction endonuclease genes is to develop a simple and reliable method to identify such clones within complex 'libraries', i.e. populations of clones derived by 'shotgun' procedures, when they occur at frequencies as low as 10 ⁇ 3 to 10 ⁇ 4.
the method should be selective, such that the unwanted, majority, of clones are destroyed while the desirable, rare, clones survive.
Type II restriction-modification systems are being cloned with increasing frequency.
the first cloned systems used bacteriophage infection as a means of identifying or selecting restriction endonuclease clones (HhaII: Mann et al ., Gene 3: 97-112, (1978); EcoRII: Kosykh et al ., Molec. gen. Genet 178: 717-719, (1980); PstI: Walder et al ., Proc. Nat. Acad. Sci. USA 78 1503-1507, (1981)).
a third approach and one that is being used to clone a growing number of systems, involves selecting for an active methylase gene; see for example U.S. Patent application Ser. No. 707079, (equivalent to EP 0193413A).
a potential obstacle to cloning restriction-modification genes lies in trying to introduce the endonuclease gene into a host not already protected by modification. If the methylase gene and endonuclease gene are introduced together as a single clone, the methylase must protectively modify the host DNA before the endonuclease has the opportunity to cleave it. On occasion, therefore, it might only be possible to clone the genes sequentially, methylase first then endonuclease. Another obstacle to cloning.
restriction- modification systems lies in the discovery that some strains of E.coli react adversely to cytosine modification; they possess systems that destroy DNA containing methylated cytosine (Raleigh and Wilson, Proc. Natl. Acad. Sci., USA 83:9070-9074, (1986)). Cytosine-specific methylase genes cannot be cloned easily into these strains, either on their own, or together with their corresponding endonuclease genes. To avoid this problem it is necessary to use mutant strains of E.coli (McrA ⁇ and McrB ⁇ ) in which these systems are defective.
the present invention relates to the type II restriction endonuclease NlaIV, which is obtainable from Neisseria lactamica .
NlaIV recognizes the DNA sequence 5′ GGNNCC 3′ and cleaves in the middle of the sequence between the two non-specified nucleotides to produce blunt ends; GGN/NCC (Qiang and Schildkraut, Nucleic Acids Res . 14: 1991-1999, (1986), the disclosure of which is hereby incorporated by reference herein).
the preferred method for cloning this enzyme comprises forming a library containing the DNA from N. lactamica (NRCC 2118), isolating those clones which contain DNA coding for the NlaIV modification methylase and screening among these to identify those that also contain the NlaIV restriction endonuclease gene.
the present invention relates to clones of the NlaIV restriction and modification genes, as well to the restriction endonuclease NlaIV produced from such clones.
the NlaIV genes are cloned by a method which takes advantage of the fact that certain clones which are selected on the basis of containing and expressing the NlaIV modification methylase gene also contain the NlaIV restriction gene.
the DNA of such clones is resistant to digestion, in vitro , by the NlaIV restriction endonuclease. This resistance to digestion affords a means for selectively isolating clones encoding the NlaIV methylase and restriction endonuclease.

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Life Sciences & Earth Sciences (AREA)
Health & Medical Sciences (AREA)
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Wood Science & Technology (AREA)
Engineering & Computer Science (AREA)
Bioinformatics & Cheminformatics (AREA)
Organic Chemistry (AREA)
Zoology (AREA)
Genetics & Genomics (AREA)
Molecular Biology (AREA)
Medicinal Chemistry (AREA)
Microbiology (AREA)
Biotechnology (AREA)
Biochemistry (AREA)
General Engineering & Computer Science (AREA)
General Health & Medical Sciences (AREA)
Biomedical Technology (AREA)
Enzymes And Modification Thereof (AREA)
Micro-Organisms Or Cultivation Processes Thereof (AREA)

EP89307206A 1988-07-28 1989-07-17 Procédé de production de l'endonucléase de restriction et de la méthylase Nla IV Ceased EP0355981A3 (fr)

Applications Claiming Priority (2)

Application Number	Priority Date	Filing Date	Title
US07/225,246 US5075232A (en)	1988-07-28	1988-07-28	Method for producing the nlavi restriction endonuclease and methylase
US225246		1988-07-28

Publications (2)

Publication Number	Publication Date
EP0355981A2 true EP0355981A2 (fr)	1990-02-28
EP0355981A3 EP0355981A3 (fr)	1990-03-07

Family

ID=22844132

Family Applications (1)

Application Number	Title	Priority Date	Filing Date
EP89307206A Ceased EP0355981A3 (fr)	1988-07-28	1989-07-17	Procédé de production de l'endonucléase de restriction et de la méthylase Nla IV

Country Status (4)

Country	Link
US (1)	US5075232A (fr)
EP (1)	EP0355981A3 (fr)
JP (1)	JPH02167074A (fr)
DE (1)	DE355981T1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number	Priority date	Publication date	Assignee	Title
EP0477532A1 (fr) *	1990-08-30	1992-04-01	New England Biolabs, Inc.	Fragment d'ADN codant pour l'endonucléase de restriction et la méthylase Nla III.

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number	Priority date	Publication date	Assignee	Title
US5200337A (en) *	1991-10-25	1993-04-06	New England Biolabs, Inc.	Type ii restriction endonuclease, apo i, obtainable from arthrobacter protophormiae and a process for producing the same
CA2159081C (fr) *	1993-03-24	2000-11-21	David Mead	Preparations d'endonuclease de restriction de type dinucleotide, et methodes d'utilisation
EP2568040A1 (fr)	2005-08-04	2013-03-13	New England Biolabs, Inc.	Nouvelles endonucléases de restriction, ADN codant ces endonucléases et procédés pour identifier de nouvelles endonucleases avec la spécificité identique ou variée

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number	Priority date	Publication date	Assignee	Title
IN166864B (fr) *	1985-03-01	1990-07-28	New England Biolabs Inc

1988
- 1988-07-28 US US07/225,246 patent/US5075232A/en not_active Expired - Lifetime
1989
- 1989-07-17 DE DE89307206T patent/DE355981T1/de active Pending
- 1989-07-17 EP EP89307206A patent/EP0355981A3/fr not_active Ceased
- 1989-07-28 JP JP1196423A patent/JPH02167074A/ja active Pending

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number	Priority date	Publication date	Assignee	Title
EP0477532A1 (fr) *	1990-08-30	1992-04-01	New England Biolabs, Inc.	Fragment d'ADN codant pour l'endonucléase de restriction et la méthylase Nla III.
US5278060A (en) *	1990-08-30	1994-01-11	New England Biolabs, Inc.	Method for producing the Nla III restriction endonuclease and methylase

Also Published As

Publication number	Publication date
EP0355981A3 (fr)	1990-03-07
US5075232A (en)	1991-12-24
DE355981T1 (de)	1994-02-03
JPH02167074A (ja)	1990-06-27

Legal Events

Date	Code	Title	Description
1990-01-13	PUAI	Public reference made under article 153(3) epc to a published international application that has entered the european phase	Free format text: ORIGINAL CODE: 0009012
1990-01-17	PUAL	Search report despatched	Free format text: ORIGINAL CODE: 0009013
1990-02-28	AK	Designated contracting states	Kind code of ref document: A2 Designated state(s): DE FR GB
1990-03-07	AK	Designated contracting states	Kind code of ref document: A3 Designated state(s): DE FR GB
1990-10-31	17P	Request for examination filed	Effective date: 19900906
1992-08-19	17Q	First examination report despatched	Effective date: 19920708
1993-10-01	EL	Fr: translation of claims filed
1994-02-03	DET	De: translation of patent claims
1995-09-19	STAA	Information on the status of an ep patent application or granted ep patent	Free format text: STATUS: THE APPLICATION HAS BEEN REFUSED
1995-11-08	18R	Application refused	Effective date: 19950623

Publication	Publication Date	Title
US5002882A (en)	1991-03-26	Method for producing the XmaI restriction endonuclease and methylase
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EP0321271B1 (fr)	1994-03-23	Procédé de production de l'endonucléase de restriction et de la méthylase HgiAI
US4983522A (en)	1991-01-08	Method for producing the HinPI restriction endonuclease and methylase
US5053330A (en)	1991-10-01	Method for producing the mwoi restriction endonuclease and methylase
US4988620A (en)	1991-01-29	Method for producing the FnuDI restriction endonuclease and methylase
EP0332406B1 (fr)	1994-02-16	Procédé de production de l'endonucléase de restriction et de la méthylase Ase I
US5075232A (en)	1991-12-24	Method for producing the nlavi restriction endonuclease and methylase
US5030569A (en)	1991-07-09	Method for producing the AFL II restriction endonuclease and methylase
EP0321267B1 (fr)	1995-02-22	Procédé de production de l'endonucléase de restriction et de la méthylase AccI
EP0321268A2 (fr)	1989-06-21	Procédé de production de l'endonucléase de restriction et de la méthylase Ban I
US5298404A (en)	1994-03-29	Method for producing the Hpa I restriction endonuclease and methylase
EP0314346B1 (fr)	1995-08-30	Procédé de préparation de l'endonucléase de restriction Afl II
ENDONUCLEASE	0	Looney et al.[45] Date of Patent: Mar. 12, 1991
ENDONUCLEASE	1991	Barsomian et al.
ENDONUCLEASE	0	Van Cott et al.[45] Date of Patent: Jan. 29, 1991
ENDONUCLEASE	0	Barsomian et al.[45] Date of Patent: Mar. 12, 1991